pISSN 1229-845X, eISSN 1976-555X JOURNAL OF J. Vet. Sci. (2010), 11(4), 359-361 DOI: 10.4142/jvs.2010.11.4.359 Veterinary Received: 05 Feb. 2010, Accepted: 28 May 2010 Science Case Report Elevated fructosamine concentrations caused by IgA paraproteinemia in two dogs

Florian Zeugswetter1,*, Miriam Kleiter1, Birgitt Wolfesberger1, Ilse Schwendenwein2, Ingrid Miller3

Departments of 1Companion Animals and Horses, 2Pathobiology, and 3Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria

An 8-year-old male Austrian Pinscher and a 14-year-old male Because hyperglobulinemia commonly leads to Golden Retriever were presented for evaluation due to compensatory hypoalbuminemia, fructosamine values are unexplainable high fructosamine values despite euglycemia usually low in markedly hyperglobulinemic patients. and epistaxis in combination with polydipsia/polyuria, However, exceptional cases of unknown cause have been respectively. Blood analysis revealed severe hyperglobulinemia, reported [9]. Here, markedly elevated fructosamine hypoalbuminemia and markedly elevated fructosamine concentrations in two nondiabetic dogs with a monoclonal concentrations in both dogs. Multiple myeloma with IgA- IgA-gammopathy are described. monoclonal gammopathy was diagnosed by serum and urine electrophoresis including immunodetection with an anti-dog Case No. 1 IgA antibody and bone marrow aspirations. Diabetes mellitus An 8-year-old 21 kg male Pinscher was presented with a was excluded by repeated plasma and urine glucose 2-week history of vague gastrointestinal signs (inconsistent measurements. Fructosamine values were positively correlated appetite, flatulence) and lethargy. An extended health check with globulin, but negatively correlated with albumin up-profile (IDEXX Vet Med Labor, Germany) revealed concentrations. These cases suggest that, as in human patients, unexplainable high fructosamine concentrations as determined monoclonal IgA gammopathy should be considered as a based on a colorimetric test conducted using a Roche Hitachi possible differential diagnosis for dogs with high fructosamine 91 Chemistry Analyzer (Boehringer, Germany; Table 1), concentrations. despite euglycemia (5.2 mmol/L, reference range 3-5.6 Keywords: dogs, fructosamine, monoclonal IgA, paraproteinemia Table 1 . Fructosamine and protein concentration of two dogs with IgA-monoclonal gammopathy before and during chemotherapy

Fructosamines are glycated proteins that are measured to Days of Fructosamine Total protein Albumin Globulin identify diabetes mellitus and as indicators of glycemic therapy (μmol/L) (g/dL) (g/dL) (g/dL) control [4,9]. Their concentration is not influenced by acute hyperglycemia [6], but misleading elevations are possible Case 1 in hypothyroid patients [8]. Because fructosamines are the 0 654 10.54 1.60 8.90 product of the spontaneous condensation of glucose with 10 583 10.67 1.47 9.20 primary amines followed by Amadori rearrangement, their 24 519 9.77 1.93 7.84 concentrations also depend on protein concentration, turnover 116 447 8.59 2.49 6.10 and composition [4]. The specific proteins involved are 383 307 8.43 2.72 5.71 currently not well established, but there is a great deal of evidence that albumin is a major contributor. Studies Case 2 conducted in dogs identified positive correlations between 0 506 11.02 1.94 9.98 albumin and fructosamine, but little or no correlation 7 398 9.22 1.86 7.36 between total protein and fructosamine concentration [4,9]. 19 243 6.16 2.37 3.78 128 146 7.38 2.62 4.76 *Corresponding author Tel: +43-1-25077-5101; Fax: +43-1-25077-5190 Reference ＜ 370 6 to 7.5 2.6 to 4.7 1.5 to 3.5 E-mail: [email protected] range 360 Florian Zeugswetter et al. mmol/L) and normal serum thyroxine values (24.5 nmol/L, Austria) and 0.5 mg/kg prednisolone (Nycomed; Nycomed, reference range 19.3-58 nmol/L; measured using a DPC Austria) SID. After ten days, the melphalan was reduced to Immulite 1000; Siemens, USA). Severe hyperglobulinemia 0.05 mg/kg SID and prednisolone was reduced to 0.5 mg/kg associated with hypoalbuminemia was also detected (Table EOD. Upon treatment, the well-being of the dog clearly 1). The differential diagnosis for hyperglobulinemia in the improved and changes in the albumin and IgA levels were absence of hyperalbuminemia included polyclonal (chronic observed in the electrophoretic patterns. Specifically, the IgA inflammation or infection) and monoclonal (lymphoid concentrations decreased and the albumin band became tumors) gammopathy. The dog was found to be negative for more prominent (both seen as changes in the thickness and leishmania antibodies (immunofluorescence), ehrlichiosis intensity of the respective bands in Fig. 2, Lanes 4-9). The and anaplasmosis (PCR). No osteolytic lesions or lung metastasis were observed upon radiographic examinations. A bone marrow needle aspiration from the ileum crest was conducted. Due to cluster formation, an exact cell count of the bone marrow aspirate could not be obtained, but plasma cells likely represented ＞ 30% of all nucleated cells. Routine serum electrophoresis on cellulose acetate strips (Interlab Genio Electrophoresis-System, Densitometric Scanning, Elfolab Software; Menarini Diagnostics, Austria) displayed a large peak in the beta/gamma globulin region, comprising about 70% of the overall serum protein (Fig. 1). An additional SDS-PAGE conducted under reducing conditions [5] revealed a monoclonal gammopathy of IgA class (Fig. 2), evidenced by a prominent band at 59 kD (the α- = heavy chain) and a very narrow and distinct light chain of 28 kD. In contrast, Fig. 2. Serum protein pattern of a dog (Case 1) with monoclonal gammopathy as determined by SDS-PAGE (8 × 6 cm gels with 10- light chains of polyclonal immunoglobulins display much 15% polyacrylamide gradient) and detection of the immunoglobulin larger heterogeneity and thus a much broader band, as seen class. General protein staining (silverstain). Samples from time in the control sample (Lane 3 of Fig. 2) or in IgG preparations course (before and during therapy), run under reducing conditions: (Lane 2 of Fig. 2) [7]. The immunoglobulin class was further Lane 1: Molecular weight standard, Lane 2: Dog IgG pure substance, Lane 3: Serum of a healthy dog, Lanes 4-9: Serum confirmed in an immunoblot with an anti-dog IgA antibody samples from time course, at: 23 days before therapy and on the (α-chain specific; Bethyl Laboratories, USA). Small 10th, 24th, 50th, 69th, and 88th day of therapy; samples of this amounts of monoclonal antibody could also be detected in patient were more dilute than the healthy control. SA: serum albumin; MAb: monoclonal antibody (IgA class, consisting of the urine. The diagnosis of IgA-multiple myeloma was based heavy (α-) and light chains (L-)). on the increased serum concentration of monoclonal immunoglobulins, proteinuria and dominance of plasma cells in the bone marrow. Oral therapy was started with 0.1 mg/kg melphalan hydrochloride (Alkeran; GlaxoSmithKline,

Fig. 3. Regression of serum fructosamine and total serum protein, Fig. 1. Routine serum electrophoresis on a cellulose acetate strip globulin and albumin concentrations of a dog (Case 1) with IgA with plastic support of a dog (Case 1) conducted using an paraproteinemia before and during therapy. The lines represent automated system. the regression lines. Fructosamine concentration in dogs with IgA paraproteinemia 361 decrease in IgA was associated with a decrease in negative correlation between albumin and fructosamine fructosamine concentration (Table 1, selected days). concentration in these patients. Despite the increasing Fructosamine was positively correlated (Pearson’s correlation, albumin concentrations, fructosamines decreased in 19 measurements) with total protein (r = 0.831, p ＜ 0.001) parallel to clinical and biochemical remission. In human and globulin (r = 0.888, p ＜ 0.001), but negatively correlated medicine, it is well established that elevated IgA has to be with albumin (r = 󰠏0.781, p ＜ 0.001) concentration (Fig. 3). taken into consideration when interpreting fructosamine to Repeated urine dipstick measurements were negative for avoid misinterpretations. No such effect is seen in patients glucose, and blood glucose concentrations were within the with elevated polyclonal IgA and monoclonal IgG or IgM normal range. myeloma patients [1,2]. The results from the two euglycemic dogs evaluated in Case No. 2 this study were parallel to findings in human patients that A 14-year-old male Golden Retriever was evaluated fructosamine concentrations can be markedly elevated in because of a three month history of polydipsia, polyuria, the presence of a monoclonal IgA-gammopathy. lethargy and epistaxis. Except for pale mucous membranes, Paraproteinemia should be taken into consideration in tachycardia (124 beats per min), a small palpable testical dogs with unexplained high fructosamine levels. mass and a large palpable spleen (confirmed by ultrasonography: large, dense, inhomogeneous), the clinical References examination was unremarkable. Leishmaniasis, anaplasmosis and ehrlichiosis were excluded as described in Case 1. 1. Fujita K, Curtiss LK, Sakurabayashi I, Kameko F, Radiographs of the nose did not show osteolytic lesions or Okumura N, Terasawa F, Tozuka M, Katsuyama T. foreign bodies. Platelet counts and blood coagulation times Identification and properties of glycated monoclonal IgA that were normal. Notable laboratory findings included non- affect the fructosamine assay. Clin Chem 2003, 49, 805-808. regenerative anemia (hematocrit 32%, reference range 37- 2. Fujita K, Kameko F, Kato Y, Fukushima M, Okumura N, 55%), hyperproteinemia, hypoalbuminemia (for proteins Terasawa F, Sugano M, Yamauchi K, Sato H, Kameko M, Sakurabayashi I. Mechanism of IgA-albumin complex see Table 1) and proteinuria (urinary protein/creatinine formation that affects the fructosamine assay. J Electrophor ratio, 3.95). Despite euglycemia (glucose 4.8 mmol/L) and 2006, 50, 19-23. negative urine glucose dipstick results, the fructosamine 3. Griot-Wenk ME, Busato A, Welle M, Racine BP, levels were high (Table 1). Serum thyroxine was low, but Weilenmann R, Tschudi P, Tipold A. Total serum IgE and TSH and cholesterol were within the reference range. IgA antibody levels in healthy dogs of different breeds and Multiple myeloma with IgA monoclonal gammopathy was exposed to different environments. Res Vet Sci 1999, 67, diagnosed based on the results of electrophoresis (similar to 239-243. Case 1), bone marrow biopsy (bone marrow plasmacytosis, 4. Kawamoto M, Kaneko JJ, Heusner AA, Feldman EC, ＞ 30% atypical plasma cells) and fine needle aspiration of Koizumi I. Relation of fructosamine to serum protein, the spleen (moderate extramedullary hematopoesis, high albumin, and glucose concentrations in healthy and diabetic number of atypical plasma cells). The IgA concentration dogs. Am J Vet Res 1992, 53, 851-855. 5. Laemmli UK. Cleavage of structural proteins during the was also quantified by an enzyme linked immunosorbent μ μ assembly of the head of bacteriophage T4. Nature 1970, 227, assay (IgA = 1,440 g/mL, normal range 6.1-973 g/mL; 680-685. University of Veterinary Medicine Hannover, Germany) 6. Marca MC, Loste A, Ramos JJ. Effect of acute hyper- [3]. The testicular mass was confirmed as an interstitial cell glycaemia on the serum fructosamine and blood glycated tumor. Therapy was started as described above and with the haemoglobin concentrations in canine samples. 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